Circulating cell-free DNA discriminates resolving from non-resolving thromboembolism
T Artner, T M Hofbauer, A S Ondracek, B Reil, L Engel, L Valerio, S Konstantinides, I M LangAbstract
Background
Acute pulmonary embolism resolves in the majority of cases, but sometimes, chronic thromboembolic pulmonary hypertension (CTEPH) is the consequence, and is usually recognized late. A central unmet need is a non-invasive biomarker capable of distinguishing physiological thrombus resolution from progressive, non-resolving thrombotic disease. Experimental and pathological evidence indicates that unresolved erythrocyte-rich thrombi undergo neutrophil extracellular trap (NET)–driven chronic remodeling and fibrotic transformation rather than resolution. This process may result in sustained release of neutrophil-derived cell-free DNA (cfDNA), providing a mechanism-informed basis for cfDNA as a diagnostic biomarker in CTEPH.
Methods
Circulating cfDNA was analyzed in a large, well-characterized cohort comprising healthy volunteers (HV; n = 343), patients with a history of acute pulmonary embolism without CTEPH at follow-up (PE-FU; n = 274), and patients with established CTEPH (n = 435). Total cfDNA concentration and cfDNA fragment length distributions were quantified from plasma. Ex vivo whole-blood thrombosis assays and histological analyses of acute pulmonary emboli and chronic CTEPH thrombotic material were used to link circulating cfDNA profiles to NET-driven thrombus persistence and remodeling. Group differences were assessed using Kruskal–Wallis testing with Dunn’s correction for multiple comparisons. Diagnostic performance for discriminating resolving PE from CTEPH was evaluated using receiver operating characteristic (ROC) analyses.
Results
Total circulating cfDNA concentrations normalized in patients with resolving pulmonary embolism and did not differ from healthy volunteers, whereas patients with CTEPH showed marked and selective elevation (adjusted p < 0.0001). In contrast, elongated cfDNA fragments demonstrated a graded increase across clinical states. cfDNA fragments >83 bp and >244 bp increased stepwise from healthy volunteers to PE follow-up patients and were highest in CTEPH, with all pairwise comparisons remaining significant after correction (adjusted p < 0.0001). In ROC analyses, >83 bp cfDNA achieved the highest diagnostic performance for distinguishing healthy volunteers from CTEPH (AUC = 0.95), while total cfDNA concentration provided the strongest discrimination between PE-FU patients and CTEPH (AUC = 0.89). Long cfDNA fragments >244 bp supported disease-associated persistence and impaired cfDNA degradation but showed lower diagnostic performance.
Conclusions
Circulating cfDNA integrates key biological processes underlying failed thrombus resolution and chronic thromboembolic remodeling. Total cfDNA concentration distinguishes patients with persistent thromboembolic disease after pulmonary embolism from those with physiological resolution. cfDNA-based plasma measurements may support post-PE risk stratification and guide referral for definitive diagnostic evaluation in patients with suspected CTEPH.